Differential effects of norUDCA and UDCA in obstructive cholestasis in mice

Background & Aims

The quest for effective drugs to treat cholangiopathies led to the development of norUDCA previously shown to have potent choleretic effects and to heal cholangiopathy in Abcb4 knockout (Abcb4^−/−) mice. Its mother compound UDCA had detrimental effects in common bile duct ligated (CBDL) mice, presumably related to its choleretic effects. norUDCA choleretic effects may therefore raise safety concerns when used in cholangiopathies with biliary obstruction. We therefore aimed at comparing the effects of UDCA and norUDCA in clear-cut obstructive cholestasis.

Methods

0.5% UDCA- or norUDCA-fed wild type and Abcb4^−/− mice were subjected to CBDL or selective bile duct ligation (SBDL) and compared to controls with regard to liver injury. Bile flow, bile composition, and biliary manometry were compared in UDCA-fed, norUDCA-fed and control mice. Toxicity of UDCA and norUDCA was compared in vitro.

Results

Compared to UDCA, liver injury in CBDL mice was significantly lower in almost all norUDCA groups. In SBDL mice, only UDCA induced bile infarcts in the ligated lobes, whereas norUDCA even ameliorated liver injury. In vitro, UDCA induced cellular ATP depletion and was significantly more toxic than norUDCA in HepG2 cells, mouse bile duct epithelial cells, and primary human hepatocytes.

Conclusions

Compared to norUDCA, UDCA is significantly more toxic in CBDL mice. norUDCA, in contrast to UDCA, significantly ameliorates liver injury in SBDL mice. Our findings uncover profound differences in metabolism and therapeutic mechanisms of both bile acids with important clinical consequences.

Introduction

Cholangiopathies frequently lead to biliary fibrosis and cirrhosis with the complications of end-stage liver disease [1], [2], [3], [4], [5], [6]. This group of liver diseases still represents a major indication for liver transplantation [6], [7], [8], [9], [10], [11], [12], underscoring the limited efficacy of currently available medical treatments in cholangiopathies and the urgent need for novel pharmaceutical strategies [4], [6], [13], [14]. UDCA, at present the only approved drug for PBC, appears to exert its beneficial effects by stimulation of bile flow, rendering bile composition less toxic, and reducing the retention of potentially toxic bile acids in hepatocytes and liver injury [5], [15], [16], [17], [18]. However, the efficacy of UDCA in different cholangiopathies, such as PSC and SSC, is limited [4], [6], [19]. Alan Hofmann’s elegant cholehepatic shunting concept led to the design of side chain-shortened norUDCA with substantial different physicochemical and physiological properties compared to its mother compound UDCA [20], [21], [22], [23], [24]. norUDCA was previously also shown to have superior therapeutic effects in Mdr2/Abcb4 knockout mice (Abcb4^−/−) as model for sclerosing cholangitis [Supplementary Refs. 25–27]. Moreover, norUDCA has potent cholerertic effects in rodents [Supplementary Refs. 26,27] and humans [Supplementary Ref. 28]. Therefore, norUDCA is about to undergo further clinical development for cholangiopathies.

BDL represents the extreme variant of obstructive cholestasis and a well-characterized rodent model system to study the pathophysiology of cholestatic liver disease [Supplementary Refs. 29,30]. We previously demonstrated increased liver injury with aggravation of bile infarcts in UDCA-fed CBDL mice [Supplementary Ref. 31] and postulated that this may primarily be related to increased biliary pressure due to the choleretic effects of UDCA leading to the rupture of the canals of Herring [Supplementary Ref. 31]. Consequently, ductular bile (with millimolar concentrations of bile acids) leaking into the liver parenchyma leads to bile infarcts with oncotic hepatocyte cell death. Comparable findings were obtained in UDCA-fed Abcb4^−/− mice with partial biliary obstruction as a result of sclerosing cholangitis, but Abcb4^−/− mice have even increased bile flow, and this model represents, at least from a biliary physiology point of view, no clear-cut situation with complete obstructive cholestasis [Supplementary Ref. 31]. In this model system, UDCA feeding also significantly increased the number and size of bile infarcts in a dose-dependent manner [Supplementary Ref. 31]. Our concept was further supported by amelioration of liver injury in CBDL FXR knockout (FXR^−/−) mice, which may be attributed to lower biliary pressure and a more hydrophilic bile acid pool in this genotype [Supplementary Refs. 32–34]. Consequently, CBDL FXR^−/− mice lacked bile infarcts and showed considerably reduced ductular reaction [Supplementary Ref. 32], both mainly triggered by increased biliary pressure in CBDL rodents [Supplementary Refs. 35,36]. This concept is in contrast with the beneficial effects of norUDCA in Abcb4^−/− mice despite superior choleretic effects compared to UDCA [Supplementary Refs. 26,27]. These findings may therefore challenge our initial hypothesis that aggravation of bile infarcts in UDCA-fed CBDL and Abcb4^−/− mice is primarily caused by biliary pressure [Supplementary Refs. 31,37]. Consequently, the aim of the current study was a face-to-face comparison of UDCA and norUDCA therapeutic mechanisms in the extreme variant of obstructive cholestasis with the aid of the CBDL and selective bile duct ligated (SBDL) mouse model. Since norUDCA represents a promising new drug for cholangiopathies with significant obstructive components, information on its effects in clear-cut obstructive cholestasis animal models should be of great value.